Great progresses have been recently achieved to produce chemical materials (such as ethanol, succinate etc.) by microbial fermentation. Compared to traditional petrochemical process, microbial fermentation has many advantages including high productivity, low cost, and the use of renewable raw materials instead of petrochemicals.
In microbial fermentation, E. coli is the most commonly used host for obtaining high-producing strain since it has clear physiological and genetic characteristic and can be genetically modified easily. E. coli also grows fast and can be cultured easily. Under anaerobic fermentation, E. coli generally consumes saccharides or their derivatives and produces mix-acids including formate, acetate, lactate, succinate, ethanol etc. For the strains of wild-type E. coli, the yield of ethanol and succinate is low. Recombinant DNA technology of microbial strains has been developed, which has been applied to modify specific enzymes involved in metabolic pathways of E. coli, for obtaining high-producing strains.
Pyruvate dehydrogenase complex (PDH), a complex of three enzymes, plays an important role in metabolic pathways of E. coli, catalyzing the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA with reducing NAD+ into NADH. Acetyl-CoA produced in this reaction go through tricarboxylic acid cycle (TCA) to perform cellular respiration. Pyruvate dehydrogenase complex establishes a connection between glycolysis metabolic pathway and TCA. Pyruvate decarboxylation is also called “pyruvate dehydrogenation”, because of involving oxidization of pyruvate (Hansen et al., 1996 Biochim Biophys Acta 122: 355-358; Bisswanger 1981 J Biol Chem 256: 815-822; Quail et al., 1994 J Mol Microbiol 12:95-104).
During microbial anaerobic fermentation, NAD+ and NADH are important co-factors for maintaining oxidation-reduction reactions. In this process, NAD+ is key electron acceptor, and NADH as co-factor determines the supply of reducing equivalent in electron transfer (Garrigues et al., 1997 J Bacteriol 179: 5282-5287; Cassey et al., 1998 FEMS Microbiol Lett 159:325-329). During glycolysis, one molecule glucose generates two molecules NADH, while one molecule glucose can generate two molecules acetyl-CoA by pyruvate decarboxylation, with four molecules NADH generated (glucose→2 acetyl-CoA→4 NADH). Two more molecules NADH are generated in conversing pyruvate to acetyl-CoA than that of pyruvate into formate, producing additional reducing equivalent. Therefore, the activity of pyruvate dehydrogenase (PDH) in pyruvate decarboxylation is significant for increasing the supply of reducing equivalent in metabolic pathways.
PDH is the important enzyme connecting glycolysis and TCA, and its activity is low under anaerobic conditions although is high under aerobic conditions. The activity of PDH is inhibited by the acetyl-CoA and NADH produced by PDH reaction. NADH is an important reducing equivalent for microbial cell-factories, but NADH of high concentration inhibits PDH, making it as a critical rate-limiting enzyme in metabolic pathways. Kim (Kim et al., 2008 J Bacteriol 190: 3851-3858) isolated a mutant strain E354K (lpd101), whose dihydrolipoamide dehydrogenase (LPD) of PDH was mutated, which was identified to be responsible for reducing sensitivity of PDH to NADH under anaerobic conditions, increasing ethanol production by this pathway. Zhou et al. (Zhou et al., 2008 Biotechnol Lett 30:335-342) increased PDH the activity by introducing lpd mutation and the regulating aceEF gene, increasing the biomass and the yield of stain in fermentation processes.
In order to improve the titer and/or yield of E. coli in the production of chemical materials, it is desired to further modify the metabolic pathways of E. coli. 